Thermo Fisher Scientific Precision ID mtDNA Panels Owner's manual

Brand: Thermo Fisher Scientific

Model: Precision ID mtDNA Panels

Type: Owner's manual

Precision ID mtDNA Panels with the HID Ion S5™/HID Ion

GeneStudio™ S5 System: Manual Library Preparation

Catalog Numbers A30938, A31443

Pub. No. MAN0017771 Rev. A.0

Note: For safety and biohazard guidelines, see the “Safety”

appendix in the Precision ID mtDNA Panels with the HID Ion

S5™/HID Ion GeneStudio™ S5 System Application Guide

(Pub. No. MAN0017770). Read the Safety Data Sheets (SDSs) and

follow the handling instructions. Wear appropriate protective

eyewear, clothing, and gloves.

■Extract, then quantify input DNA ................... 1

■Prepare the mtDNA target amplication reaction ....... 1

■Amplify the targets ............................... 2

■Partially digest amplicons ......................... 3

■Ligate adapters to the amplicons, then purify .......... 3

■Quantify the libraries by qPCR ..................... 4

■(Optional) Amplify and purify the libraries ............. 4

■Dilute, pool, and store the libraries ................... 6

■Limited product warranty ......................... 6

Extract, then quantify input DNA

1. Extract mtDNA using the PrepFiler Express BTA™ Forensic

DNA Extraction Kit (Cat. No. 4441351).

2. Quantify DNA using one of the recommended DNA

quantication kits that are listed in the Precision ID mtDNA

Panels with the HID Ion S5™/HID Ion GeneStudio™ S5 System

Application Guide.

Note: If you are using the Quantiler™ HP or Quantiler™

Trio DNA Quantication Kit, estimate the mtDNA input by

using 10% of the gDNA Small Amplicon (SA) quantity. For

example, for non-degraded samples use 0.1 ng of gDNA in

target amplication reactions.

IMPORTANT! If you are using the PrepFiler Express BTA™

Forensic DNA Extraction Kit to extract mtDNA from non-

BTA substrates such as blood or buccal, perform this

modication: During the lysis, incubate the column/tube

assembly at 56°C, then shake at 750 rpm for 40 minutes.

Prepare the mtDNA target ampliﬁcation reaction

1. Use the following table to choose the amplication method

that is based on your sample type.

Sample type Method Go to

Low copy number samples 2-in-1 step 2

Non-degraded samples Conservative step 3

Note: For descriptions of the methods, see the Precision ID

mtDNA Panels with the HID Ion S5™/HID Ion GeneStudio™ S5

System Application Guide (Pub. No. MAN0017770).

2. For the 2-in-1 method, prepare two master mixes, one for

each pool:

a. Prepare the master mix for Precision ID mtDNA panel

Pool 1, then add to each well of a 96-well plate:

Component Volume

5X Ion AmpliSeq™ HiFi Mix (red cap) 4 µL

Precision ID mtDNA panel Pool 1 10 µL

gDNA, 0.1 ng[1] X µL[2]

Nuclease-free Water 6 – X µL

Total 20 µL

[1] 0.1 ng ≈ 2900 mtDNA copies. gDNA quantifications were used to

extrapolate the copy number of mtDNA. If more than 0.1 ng of

gDNA is used, appropriately adjust the number of PCR cycles in

“Amplify the targets“ on page 2.

[2] ≤6 µL

b. Prepare the master mix for Precision ID mtDNA panel

Pool 2, then add to each well of a 96-well plate:

Component Volume

5X Ion AmpliSeq™ HiFi Mix (red cap) 4 µL

Precision ID mtDNA panel Pool 2 10 µL

gDNA, 0.1 ng[1] X µL[2]

Nuclease-free Water 6 – X µL

Total 20 µL

[1] 0.1 ng ≈ 2900 mtDNA copies. gDNA quantifications were used to

extrapolate the copy number of mtDNA. If more than 0.1 ng of

gDNA is used, appropriately adjust the number of PCR cycles in

“Amplify the targets“ on page 2.

[2] ≤6 µL

QUICK REFERENCE

For Research, Forensic, or Paternity Use Only. Not for use in diagnostic procedures.

c. Go to step 4.

3. For the Conservative method, prepare two master mixes, one

for each pool:

a. Prepare the master mix for Precision ID mtDNA panel

Pool 1, then add to each well of a 96-well plate:

Component Volume

5X Ion AmpliSeq™ HiFi Mix (red cap) 2 µL

Precision ID mtDNA panel Pool 1 5 µL

gDNA, 0.1 ng[1] X µL[2]

Nuclease-free Water 3– X µL

Total 10 µL

[1] 0.1 ng ≈ 2900 mtDNA copies. gDNA quantifications were used to

extrapolate the copy number of mtDNA. If more than 0.1 ng of

gDNA is used, appropriately adjust the number of PCR cycles in

“Amplify the targets“ on page 2.

[2] ≤3 µL

b. Prepare the master mix for Precision ID mtDNA panel

Pool 2, then add to each well of a 96-well plate:

Component Volume

5X Ion AmpliSeq™ HiFi Mix (red cap) 2 µL

Precision ID mtDNA panel Pool 2 5 µL

gDNA, 0.1 ng[1] X µL[2]

Nuclease-free Water 3 – X µL

Total 10 µL

[1] 0.1 ng ≈ 2900 mtDNA copies. gDNA quantifications were used to

extrapolate the copy number of mtDNA. If more than 0.1 ng of

gDNA is used, appropriately adjust the number of PCR cycles in

“Amplify the targets“.

[2] ≤3 µL

c. Go to step 4.

4. Seal the plate with a MicroAmp™ Clear Adhesive Film. To

prevent evaporation, create a tight seal by applying pressure

with an applicator.

5. Vortex the plate thoroughly, then centrifuge to collect

droplets. Place a MicroAmp™ Compression Pad on the plate,

then go to “Amplify the targets“.

Amplify the targets

The cycle number for target amplication depends on the panel

and the amount of input DNA. Cycle numbers can be increased if

the quality or quantity of input DNA is uncertain.

IMPORTANT! When amplifying multiple samples in a single PCR

plate, ensure that the input DNA across the samples is roughly

equivalent, or the PCR cycle number is based on the sample with

the lowest quantity. This ensures that the selected cycle number for

target amplication is optimal for all the samples in the run.

Cycle numbers for each panel depending on input DNA

Panel Amount of input

gDNA Number of cycles

Precision ID mtDNA

Whole Genome Panel

Precision ID mtDNA

Control Region Panel

0.1 ng

(~2900 mtDNA

copies)[1]

21 cycles

<0.1 ng 21 cycles + 1 to 5

cycles

[1] gDNA quantification was used to extrapolate the copy number of mtDNA. The

actual number of mtDNA copies varies from sample source (for example bone,

blood, saliva, hair, etc.).

1. To amplify target regions, run the following program:

Stage Step Temperatu

re Time

Hold Activate the

enzyme

99°C 2 minutes

Cycle number

(see preceding

table)

Denature 99°C 15 seconds

Anneal and

extend

60°C 4 minutes

Hold — 10°C Hold [1]

[1] Store reactions at 10°C overnight on the thermal cycler. For longer-term

storage, store covered at –20°C for up to one month.

2. Proceed using one of the following options:

Method Action

2-in-1 method Transfer 10 µL from each pool into a new

well, for a total of 20 µL. Continue the

library preparation as if you are processing

one sample. Use one barcode adapter.

Conservative

method

Transfer 10 µL from Pool 2 into the well

containing Pool 1, for a total of 20 µL.

Continue the library preparation as if you

are processing one sample. Use one

barcode adapter.

STOPPING POINT For either method, the target

amplication reactions can be stored at 10°C overnight on the

thermal cycler. For longer-term storage, store covered at

−20°C for up to one month.

Precision ID mtDNA Panels: Manual Library Preparation Quick Reference

Partially digest amplicons

1. Remove the plate seal, then add 2 µL of FuPa Reagent

(brown cap) to each amplied sample. The total volume is

~22 µL.

2. Seal the plate with a clear adhesive lm, vortex thoroughly,

then spin down to collect droplets.

3. Load in the thermal cycler, then setup and run the following

thermal cycling conditions:

Temperature Time

50°C 10 minutes

55°C 10 minutes

60°C 20 minutes

10°C Hold (for up to 1 hour)

STOPPING POINT Store the plate at –20°C.

Ligate adapters to the amplicons, then purify

You must ligate a dierent barcode to each library when:

• sequencing multiple libraries on a single chip

• sequencing multiple replicates of DNA libraries from the

same sample on a single chip

The barcode adapters included in the Precision ID IonCode™ 1−96

Kit in 96 Well PCR Plate are provided at the appropriate

concentration, and include forward and reverse adapters in a

single well. No further handling is necessary.

IMPORTANT! When handling barcode adapters, avoid cross-

contamination. After use, reseal the barcode adapter plate with

adhesive lm and store at −30°C to −5°C.

Perform the ligation reaction

IMPORTANT! If there is visible precipitate in the Switch Solution,

vortex or pipet up and down at room temperature to resuspend.

1. Carefully remove the plate seal, then add the following

components to each well containing digested amplicons in

the order listed.

IMPORTANT! Add the DNA Ligase last. Do not combine

DNA Ligase and adapters before adding to digested

amplicons.

Order of

addition Component Volume

1 Switch Solution (yellow cap) 4 µL

2 Precision ID IonCode™ Barcode

Adapter

2 µL

3 DNA Ligase (blue cap) 2 µL

—Total volume ~30 µL

Note:

·2-in-1 method: use one barcode

·Conservative method: use one barcode

2. Seal the plate with a new MicroAmp™ Clear Adhesive Film,

vortex thoroughly, then centrifuge to collect droplets.

3. Load the plate in the thermal cycler, then perform the

ligation reaction using the following temperature program:

Panel Temperature Time

Precision ID mtDNA

Whole Genome

Panel

Precision ID mtDNA

Control Region

Panel

22°C 30 minutes

68°C 10 minutes

10°C Hold (for up to 1

hour)

Purify the libraries

1. Carefully remove the plate seal, then add 45 µL (1.5X sample

volume) of Agencourt™ AMPure™ XP Reagent to each library.

2. Pipet up and down 5 times to mix the bead suspension with

the DNA thoroughly, then incubate the mixture for 5 minutes

at room temperature.

Alternatively, use a plate mixer (such as the Eppendorf™

MixMate™ mixer with the 96 × 0.2-mL PCR tube holder) to

mix the bead suspension. Seal the plate, mix for 5 minutes at

2,000 rpm at room temperature, then centrifuge the plate

briey to collect droplets.

3. Place the plate in a magnetic rack (such as the DynaMag™–96

Side Magnet; Cat. No. 12331D), then incubate for 2 minutes

or until solution clears.

4. Carefully remove, then discard the supernatant without

disturbing the pellet.

5. Add 150 µL of freshly prepared 70% ethanol, then move the

plate side-to-side in the two positions of the magnet to wash

the beads. Remove, then discard the supernatant without

disturbing the pellet.

6. Repeat step 5 for a second wash.

7. Ensure that all ethanol droplets are removed from the wells.

Keep the plate in the magnet, then air-dry the beads at room

temperature for 5 minutes.

Elute the libraries

1. Remove the plate containing the library from the magnet,

then add 50 µL of Low TE to the pellet to disperse the beads.

2. Seal the plate with a MicroAmp™ Clear Adhesive Film, then

vortex thoroughly.

Precision ID mtDNA Panels: Manual Library Preparation Quick Reference

3. Incubate for 5 minutes at room temperature, then centrifuge

to collect droplets.

IMPORTANT! For maximum recovery, ensure that the

suspension incubates for at least 5 minutes at room

temperature.

4. Place the plate on the magnet for at least 2 minutes.

STOPPING POINT Samples can be stored with beads at 4°C for up

to one month. For long-term storage at −20°C, place the plate in

the magnet, then transfer the sample supernatants to a new plate.

Do not store libraries at −20°C in the presence of beads.

Quantify the libraries by qPCR

After eluting each Precision ID library, determine concentration by

qPCR with the Ion Library TaqMan® Quantitation Kit (Cat. No.

4468802).

Dilute the libraries for quantiﬁcation

1. If samples have been stored at 4°C, vortex the plate, then

centrifuge to collect droplets.

2. Place the plate in the magnetic rack for 2 minutes, or until the

supernatant clears.

3. Prepare 1:100 dilutions by removing 2 µL of supernatant,

then combine with 198 µL of Nuclease-free Water.

4. After removing the aliquots, store the plate at 4°C.

Quantify the libraries

Use the Ion Library TaqMan® Quantitation Kit to analyze each

sample, standard, and negative control in duplicate 20-µL

reactions.

1. Prepare three 10-fold serial dilutions of the E. coli DH10B

Control Library (~68 pM; provided in the kit) at the

concentrations listed in the following table. Label them as

standards, then use these concentrations in the qPCR

experiment setup.

Standard Control Library

volume

Nuclease-free

Water volume Concentration

1 5 µL (undiluted) 45 µL 6.8 pM

2 5 µL Std 1 45 µL 0.68 pM

3 5 µL Std 2 45 µL 0.068 pM

2. Prepare sucient reaction mixture for replicate reactions for

each sample, negative control, and control library dilution.

Add an extra reaction to compensate for pipeing error. For

each reaction, combine 10 µL of Ion Library qPCR Master

Mix and 1 µL of Ion Library TaqMan® Quantitation Assay,

20X in a tube, then mix thoroughly.

Component Volume (1 reaction)

Ion Library TaqMan® qPCR Mix 10 µL

Ion Library TaqMan® Quantitation

Assay, 20X

1 µL

3. Aliquot 11 µL into each reaction well (two wells per reaction)

of a PCR plate.

4. Add 9 µL of the diluted (1:100) sample library, each control

library dilution, or negative control to reaction wells, for a

total reaction volume per well of 20 µL.

5. Set up the real-time PCR instrument.

a. Enter the concentrations of the control library

standards.

b. Select ROX™ Reference Dye as the passive reference

dye.

c. Enter a reaction volume of 20 µL.

d. Select FAM™ dye/MGB as the TaqMan® probe

reporter/quencher.

e. Enter the following run parameters, depending on your

system.

Real-time PCR

System Stage Temperature Time

7500 Real-Time

PCR Instrument

with SDS Software

v1.2.3

Hold 50°C 2 minutes

Hold 95°C 20 seconds

40 Cycles

95°C 3 seconds

60°C 32 seconds

7500 Real-Time

PCR Instrument

with HID Real-Time

PCR Analysis

Software v1.1 or

v1.2

Hold 50°C 2 minutes

Hold 95°C 20 seconds

40 Cycles

95°C 3 seconds

60°C 30 seconds

6. Run the reactions, then collect the real-time data.

See for library concentrations required for template preparation.

Depending on your quantication results, proceed with one of the

following options:

• If sucient library was prepared, continue to .

• If insucient library was prepared, continue to “(Optional)

Amplify and purify the libraries“.

• Continue with less than optimal library concentration. See

"Troubleshooting" in the Precision ID mtDNA Panels with the

HID Ion S5™/HID Ion GeneStudio™ S5 System Application Guide

(Pub. No. MAN0017770) for eects of using low library

concentration.

(Optional)

Amplify and purify the libraries

A library that yields less than the recommended concentration can

be rescued by library amplication. See the Precision ID mtDNA

Panels with the HID Ion S5™/HID Ion GeneStudio™ S5 System

Application Guide (Pub. No. MAN0017770) for the full procedure.

Precision ID mtDNA Panels: Manual Library Preparation Quick Reference

Amplify the libraries

1. Combine 25 µL of each unamplied library (total undiluted

library is ~50 µL, from “Elute the libraries“ on page 3) with

72 µL of Platinum™ PCR SuperMix HiFi and 3 µL of Library

Amplication Primer Mix from the Precision ID Library Kit

in one well of a 96-well PCR plate.

2. Seal the plate with MicroAmp™ Adhesive Film, vortex

thoroughly, then centrifuge briey to collect droplets.

3. Load the plate in a thermal cycler, then run the following

program:

Stage Temperature Time

Hold 98°C 2 minutes

5−10 cycles 98°C 15 seconds

64°C 1 minute

Hold 10°C Hold (for up to 24

hours)

STOPPING POINT Samples can be held overnight or up to 24

hours at 10°C on the thermal cycler. For longer periods, store

at −20°C.

Purify the ampliﬁed libraries

Perform a two-round purication process with the Agencourt™

AMPure™ XP Reagent:

•First round at 0.5X bead-to-sample-volume ratio: High

molecular-weight DNA is bound to beads, while amplicons

and primers remain in solution. Save the supernatant.

•Second round at 1.2X bead-to-original-sample-volume ratio:

Amplicons are bound to beads, and primers remain in

solution. Save the bead pellet, and elute the amplicons from

the beads.

First-round puriﬁcation

1. Tap the plate gently on a hard at surface, or centrifuge

briey to collect the contents at the boom of the wells, then

remove the plate seal.

2. Add 50 µL (0.5X sample volume) of Agencourt™ AMPure™

XP Reagent to each plate well containing ~100 µL of sample.

Mix the bead suspension with the DNA thoroughly by

pipeing up and down 5 times.

3. Incubate the mixture for 5 minutes at room temperature.

4. Place the plate in a magnet such as the DynaMag™–96 Side

Magnet for at least 5 minutes, or until the solution is clear.

5. Carefully transfer the supernatant from each well to a new

well of the 96-well PCR plate without disturbing the pellet.

IMPORTANT! The supernatant contains the desired

amplicons. Do not discard!

Second-round puriﬁcation

1. To the supernatant from “First-round purication“ on page 5

above, add 120 µL (1.2X original sample volume) of

Agencourt™ AMPure™ XP Reagent. Mix the bead suspension

with the DNA thoroughly by pipeing up and down 5 times.

2. Incubate the mixture for 5 minutes at room temperature.

3. Place the plate in the magnet for 3 minutes or until the

solution is clear. Carefully remove, then discard the

supernatant without disturbing the pellet.

IMPORTANT! The amplicons are bound to the beads. Save

the bead pellet.

4. Add 150 µL of freshly prepared 70% ethanol to each well,

then move the plate side to side in the magnet to wash the

beads. Remove and discard the supernatant without

disturbing the pellet.

5. Repeat step 4 for a second wash.

6. Ensure that all ethanol droplets are removed from the wells.

Keeping the plate in the magnet, air-dry the beads at room

temperature for 2−5 minutes. Do not overdry.

7. Remove the plate from the magnet, then add 50 µL of Low

TE to the pellet to disperse the beads.

8. Seal the plate with MicroAmp™ Adhesive Film, vortex

thoroughly, then centrifuge to collect droplets.

9. Incubate at room temperature for at least 2 minutes.

10. Place the plate in the magnet for at least 2 minutes, then

analyze an aliquot of the supernatant as described in

“Quantify the libraries by qPCR“ on page 4.

IMPORTANT! The supernatant contains the desired

amplicons. Do not discard!

Precision ID mtDNA Panels: Manual Library Preparation Quick Reference

Dilute, pool, and store the libraries

Dilute the libraries

1. After the run is complete, calculate the average concentration

of each undiluted library using the following equation:

Avg concentration of undiluted library = (qPCR quantity

mean) × (library dilution)

For example:

• qPCR quantities mean: 3 pM

• Sample library dilution: 100

The average concentration of the undiluted library:

(3 pM) × (100) = 300 pM

2. Dilute libraries as described in the following table.

Recommended library dilutions for the Ion Chef™ System

Panel Dilute to Minimum

volume

Templating size in

Planned Run

setup

Precision ID

mtDNA Whole

Genome Panel

Precision ID

mtDNA Control

Region Panel

30 pM 25 µL 200 bp

(Optional)

Pool the libraries

After diluting the sample library to its target concentration (pM),

pool equal volumes of multiple diluted libraries. Use the pooled

libraries in template preparation reactions on the Ion Chef™

Instrument.

Use the following recommendations for the number of manually-

prepared sample libraries loaded per chip. The recommendations

are based on full mitochondrial genome coverage for whole

genome, and with at least 100X coverage. You may need to adjust

the number of samples per chip based on your individual

coverage requirements, sample quality, and throughput.

Panel

Samples per Ion S5™ Chip

Ion 510™ Chip[1] Ion 520™ Chip Ion 530™ Chip[2]

Precision ID

mtDNA Control

Region Panel

37 56 —

Precision ID

mtDNA Whole

Genome Panel

— 25 32

[1] Ion 510™ Chip not supported with Precision ID mtDNA Whole Genome Panel.

[2] Ion 530™ Chip not supported with Precision ID mtDNA Control Region Panel.

Store the libraries

Store both diluted and undiluted libraries at 2°C to 8°C for up to

1 month. For long-term storage, store libraries at –30°C to −10°C.

Limited product warranty

Life Technologies Corporation and/or its aliate(s) warrant their

products as set forth in the Life Technologies' General Terms and

Conditions of Sale at www.thermosher.com/us/en/home/global/

terms-and-conditions.html. If you have any questions, please

contact Life Technologies at www.thermosher.com/support.

Manufacturer: Life Technologies Corporation | 5781 Van Allen Way | Carlsbad, CA 92008

The information in this guide is subject to change without notice.

DISCLAIMER: TO THE EXTENT ALLOWED BY LAW, THERMO FISHER SCIENTIFIC INC. AND/OR ITS AFFILIATE(S) WILL NOT BE LIABLE FOR SPECIAL, INCIDENTAL, INDIRECT,

PUNITIVE, MULTIPLE, OR CONSEQUENTIAL DAMAGES IN CONNECTION WITH OR ARISING FROM THIS DOCUMENT, INCLUDING YOUR USE OF IT.

Revision history: Pub. No. MAN0017771

Revision Date Description

A.0 9 October 2018 New Quick Reference

Important Licensing Information: These products may be covered by one or more Limited Use Label Licenses. By use of these products, you accept the terms and conditions of all

applicable Limited Use Label Licenses.

registered trademark of Roche Molecular Systems, Inc., used under permission and license. Agencourt and AMPure are trademarks of Beckman Coulter, Inc. Eppendorf, and

MixMate are trademarks of Eppendorf AG.

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9 October 2018